RU2102763C1 - Set for detection of tuberculosis mycobacteria by fluorescence method - Google Patents

Abstract

FIELD: microbiology, medicine. SUBSTANCE: set consists of staining mixture including auramine of laser purity and rhodamine C or rhodamine 4C of laser purity taken at mass ratio = (12:1)-(25:1), respectively, and background suppressing agent which includes phenoxazine dye - 3,6-diethylaminophenoxazonium perchlorate or 3,6-diethylaminophenoxazonium zinc salt, or 5-amino-9-diethylaminobenzo[a] phenoxazonium acetate, or 5-amino-9-diethylaminobenzo[a]phenoxazonium isobutyrate of laser purity. Diagnostical set ensures to detect small and ultrasmall mycobacterium forms, cell division of both typical and morphologically alternated mycobacteria. EFFECT: enhanced effectiveness of set. 1 tbl

Description

 The invention relates to microbiology. It can be used in scientific research, as well as in the practice of healthcare, veterinary medicine to detect mycobacterium tuberculosis.

Known fluorescent dyes for the diagnosis of acid-resistant mycobacteria and including the following fluorochromes and background dampers: Auramine O - potassium permanganate [1] Auramine O acridine orange [2] Auramine O - Rhodamine B potassium permanganate [3]
Prototype: the closest to the proposed complex of reagents for staining mycobacteria according to HEU [4] an aqueous solution of a coloring mixture, including auramine O
rhodamine C and an aqueous solution of a methylene blue background quencher.

 The complexes of fluorochromes mentioned above as analogues and prototype are suitable for detecting typical mycobacteria, however, they have the following disadvantages. Analogs [1, 2, 3] are characterized by insufficient selectivity. Due to the fact that under conditions of intensive chemotherapy and other influences, mycobacteria undergo significant morphological and metabolic changes, many rod-shaped microbial cells do not adsorb fluorochromes sufficiently or become discolored upon differentiation with a mixture of alcohol and hydrochloric acid. In addition, granular forms, individual L-variants of mycobacteria do not adsorb traditionally used fluorochromes to the extent that they provide microscopic and microphotographic detection.

 The disadvantages of the prototype include: the inability to detect ultrafine (<0.2 μm) forms of mycobacteria cells. In addition, methylene blue (KDM) used in the prototype as a background quencher reduces the specific glow, does not provide a contrast image of non-tuberculous microorganisms of the fungal flora of bronchial epithelial cells.

 The purpose of the invention is the creation of a set of dyes that can increase selectivity, specific luminescence, ensure reliable detection of morphologically altered forms (granular, ultrafine, L-forms), as well as detect fine-grained pathogen elements in the early stages of normal division of mycobacterial cells.

This goal is achieved by using a set of dyes containing laser-grade auramine and rhodamine C as a coloring mixture or laser-grade auramine and rhodamine 4C, and the following phenoxazine dyes are used as a background quencher: 3,6-diethylaminophenoxazonium perchlorate (oxazine-1 perchlorate , OH-120) or the zinc salt of 3,6-diethylaminophenoxazonium (oxazin-1 zinc salt, OH-121) or 5-amino-9-diethylaminobenzo / a acetate / phenoxazonium (Nile blue acetate, OH-111) or 5- isobutyrate amino-9-diethylaminobenzo / a / phenoxazonium (Nile blue isobutyrate, OH-112) of laser purity in the form of aqueous solutions at the following component ratios, g / l:
Auramine 0.5 5.0
Rhodamine C or 4C 0.02 0.4
Distilled water The rest is up to 1 liter
Background quencher: oxazine-1 perchlorate or oxazine-1 zinc salt, or Nile blue acetate, or Nile blue isobutyrate 0.01 0.6
Distilled water The rest is up to 1 liter
Along with the usual requirements for chemical purity, the proposed dyes were presented with the “laser purity” requirements adopted for luminescent generating dyes [5]. The dyes were used to control the content of impurities absorbing in the luminescence region by measuring the molar extinction coefficient at a wavelength fixed for each dye ( ε j) similar _to that described in [5]
Auramine (MA-1) was obtained by fusion of 80 g of zinc chloride, 80 g of ammonium chloride, 80 g of Michler ketone at 200 ^° C and constant stirring for 24 hours. The melt was cooled, triturated, stirred with 800 ml of water, filtered. The dye precipitated from the filtrate after salting out with sodium chloride was filtered and dried. Auramin of laser purity has spectral characteristics in ethanol: λ $\genfrac{}{}{0ex}{}{\text{gjuk \&}}{\text{Max.}}$ 431 ± 3 nm; ε ₄₃₁ (0.30 ± 0.03) • 10 ⁵ l / mol • cm; ε ₅₀₀ no more than 50 l / mol • cm.

Rhodamine C (PH-55) and rhodamine 4C (PH-56) were obtained from the corresponding technical dyes by purification by reprecipitation from a water-alcohol solution with hydrochloric acid. Rhodamine C and rhodamine 4C of laser purity have in ethanol: λ $\genfrac{}{}{0ex}{}{\text{gjuk \&}}{\text{Max.}}$ 554 ± 3 nm; ε ₅₅₄ (1.10 ± 0.15) • 10 ⁵ l / mol • cm; ε ₆₃₀ no more than 40 l / mol • cm.

5-amino-9-diethylaminobenzo / a / phenoxazonium acetate (Nile blue acetate, OH-111) was prepared from 0.5 g of Nile blue base in 50 ml of toluene with the addition of 0.1 g of acetic acid. The solution was cooled, the precipitated dye was filtered off, washed with petroleum ether, and dried. 5-amino-9-diethylaminobenzo / a / phenoxazonium isobutyrate (Nile blue isobutyrate, OH-112) was prepared similarly from 0.2 g of Nile blue base and 0.05 g of isobutyric acid. Nile blue acetate and Nile blue isobutyrate of laser purity have in ethanol: λ $\genfrac{}{}{0ex}{}{\text{sweep}}{\text{Max.}}$ 628 ± 4 nm; ε ₆₂₈ (0.651 ± 0.03) • 10 ⁵ l / mol • cm; ε ₇₃₀ no more than 65 l / mol • cm.

3,6-Diethylaminophenoxazonium perchlorate (oxazin-1 perchlorate, OH-120) and the 3,6-diethylaminophenoxazonium perchlorate (oxazin-1 zinc salt, OH-121) were obtained from 5.4 g of m-diethylaminoanisole, 6.3 ml of conc . hydrochloric acid and 6.3 ml of alcohol. To this mixture, with stirring and cooling with ice and salt to 0-3 ^° C, a solution of 3.2 g of sodium nitrate in 6.3 ml of water was added, stirred for 1 h and 4.3 g of m-diethylaminophenol in 8 were added portionwise to a boiling solution. 6 ml of alcohol, stirred for 15 minutes, 8 ml of perchloric acid (for OH-120) or 8 g of zinc chloride (for OH-121) were added, the precipitated dye was filtered off and then purified by recrystallization from ethanol. Oxazin-1 perchlorate and oxazin-1 zinc salt of laser purity have in ethanol: λ $\genfrac{}{}{0ex}{}{\text{sweep}}{\text{Max.}}$ 645 ± 4 nm; ε ₆₄₅ (1.20 ± 0.15) • 10 ⁵ l / mol • cm; ε ₇₅₀ no more than 300 l / mol • cm.

 The invention is illustrated by examples and table.

 Example 1

 Prepare 2 aqueous solutions of dyes. The first solution is an aqueous solution of a coloring mixture of auramine and rhodamine C, containing 5 g / l of the first of them and 0.4 g / l of the second dye. The second solution contains 0.6 g of oxazine-1 zinc salt (OH-121) in 1 liter of water (table, example 1).

Smears (75 ^o C, 2 h), fixed in a dry oven, are poured with a solution of the coloring mixture auramine-rhodamine for 30 minutes. Then it is washed with distilled water, decolorized with 3% hydrochloric acid alcohol for 3-5 minutes, and stained with a solution of a background quencher of oxazine for 10-15 seconds. The smears are dried, microscopted under ultraviolet light under a 40 ^x objective microscope, 4 ^x or 5 ^x eyepieces. Mycobacterium tuberculosis and their granular and ultrafine variants are stained in orange-ruby color, spherical L-shaped variants are stained in orange-yellow color.

 When processing the diagnostic material with alkalis and alkaline detergents, 0.1 ml of a 5% hydrochloric acid solution is added to 1 ml of inoculum to equalize the pH. When processing the diagnostic material with a 1% hydrochloric acid solution, 0.1 ml of a 40% KOH solution is added to 1 ml of inoculum to equalize the pH.

 Examples 2 19. Aqueous solutions were prepared in a similar manner and tested according to a similar procedure to Example 1. The composition of the components, their ratio and test results are presented in the table.

 As can be seen from the table, the use of aqueous solutions of a coloring mixture consisting of auramine and rhodamine C or auramine and rhodamine 4C of laser purity, and as a quencher, the use of aqueous solutions of zinc salt of oxazine-1 or oxazine-1 perchlorate, or Nile blue acetate, or isobutyrate of Nile blue laser purity in the presented ratios (examples 1–18) make it possible to detect small and ultrafine forms of tuberculosis mycobacteria, as well as to detect microbial cell division, in comparison with the prototype (example 19).

 Thus, the proposed set of dyes for the diagnosis of tuberculosis makes it possible to detect small and ultrafine (<0.2 μm) forms of mycobacteria, detect microbial cell division, significantly expand the capabilities of the fluorescent method for detecting not only typical but also morphologically altered mycobacteria, and create optimal microscopic possibilities and microphotographs of the causative agent of tuberculosis.

Sources of information
1. Blaiz EB Weiser OL Tull AH Mycrobacteriology Laboratory Methods. Lab. Report. US Army Medical Research and Nutrion Laboratory. Denver 1969. 235 P.

 2. Smithwick R.W. David H.L. Acridin orange as a fluorescent counterstein with the auramine acid-fast stein. Tubercle. 1971. N 52. P. 226 - 231.

 3. Traunt I.P. Brett W.A. Thomas W. Fluorescence microscopy of rtubercle bacilli steined with auramine and rhodamine. Henry Ford Hosp. Med. Bull. 1962. N 10. P. 3287 296.

 4. Modern methods of laboratory diagnosis of tuberculosis. Guidelines. M. 1992, p. 7 9.

 5. Dudkin V.S. Kogan B.Ya. Galov A.P. Savvina L.P. Thesis Doc. III All-Union. conf. "Lasers based on complex organic compounds and their application." Uzhhorod, 1980, p. 177 179.

Claims (1)

 A kit for fluorescent detection of tuberculosis mycobacteria, including a coloring mixture of auramine and rhodamine and a background quencher, characterized in that the coloring mixture contains laser-grade auramine, rhodamine C or rhodamine 4C laser-grade, taken in a mass ratio of 12 1 25 1, respectively, and 3,6-diethylaminophenoxazonium perchlorate, or 3,6-diethylaminophenoxazonium zinc salt, or 5-amino-9-diethylaminobenzo (a) phenoxazonium acetate, or 5-amino-9-diethylaminobenzo (a) isobutyrate Laser oksazoniya purity.

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